Plant disease constitutes a major and ongoing threat to human food stocks and animal feed. Most crop plants are regularly exposed to one or more pathogen(s) that can cause incredible damage resulting in substantial economical losses every year. Attack by pathogens, such as viruses, bacteria, fungi, nematodes and insects is a severe economic problem, which impacts all economically important crops, for example rice, soybean, sweet potato, wheat, potato, grape, maize and ornamental plants as cyclamen or carnation. Current protective measures rely heavily on chemical control measures for pathogens, which have undesirable environmental consequences. Natural based resistance against fungi often not exists.
A more effective approach to protecting plants from pathogen attack is to create plants that are endogenously resistant to fungi. However, plant breeders have limited sources of resistance genes against plant diseases. This can now be achieved using genetic engineering techniques, by providing the plant with genetic information required for affecting the pathogens and for being resistant to the disease caused by the pathogen. For example, in the case of a fungal pathogen, the host plant is resistant if it has the ability to inhibit or retard the growth of a fungus, the symptoms of fungal infection or the life cycle of the fungus, including its spreading. “Resistant” is the opposite of “susceptible” and may be divided into three levels:    1. Full    2. Medium    3. Partial resistance
A plant may be considered fully resistant when it shows no symptoms on infection and there is no evidence of pathogen reproduction and spreading. The host plant may be resistant to the establishment of infection, pathogen reproduction and/or pathogen spreading and transmission.
An alternative way to protect plants against pathogen infection is the generation and expression of Ab, rAb, rAb fragments and their fusions with AFPs. Pathogen-specific rAb targeted to different compartments of plant cells or different plant organs overcome many of the problems mentioned before and confer a broader spectrum of resistance to disease. To achieve this, rAb against the target proteins have to be generated by cloning the corresponding antibody heavy and light chain genes from hybridoma cells, synthetic, semi-synthetic and immunocompetent phage display, peptide display or ribosome display libraries; or by the generation of fully synthetic designer antibodies or pathogen specific peptide ligands. This is followed by subsequent modification and rAb expression in different compartments of heterologous hosts such as bacteria, yeast, algae, baculovirus infected insect cells, mammalian cells and plants. For example, antibodies and antibody-fusion proteins binding to conserved functional domains of fungal proteins or other components involved in fungal infection, growth and spreading can be used to inactivate such targets inside or outside the plant cell through immunomodulation. The feasibility of expressing recombinant antibodies for the generation of resistance has been shown recently for plant viruses (see for review Schillberg et al., 2001).
The potential of recombinant antibodies to interfere with the infection of a plant virus was demonstrated in 1993 (Tavladoraki et al., 1993). In this case, the constitutive expression of a cytosolic scFv against the coat protein of artichoke mottled crinkle virus in transgenic tobacco caused a reduction in viral infection and a delay in symptom development. This result supported the hypothesis that transgenically expressed antibodies or antibody fragments recognizing critical epitopes on structural or non-structural proteins of invading viruses may interfere with viral infection and confer viral resistance. Further support of this hypothesis was demonstrated by Voss et al. (1995). Nicotiana tabacum cv. Xanthi nc plants secreting full-size antibodies binding to intact TMV particles displayed a reduced number of necrotic local lesions when challenged with TMV. The results indicated that the number of infection events was directly correlated to the amount of secreted full-size antibodies and the local lesions were reduced by 70% when levels of apoplast targeted antibodies reached 0.23% of total soluble protein in transformed plants.
Cytosolic expression of the scFv fragment derived from this TMV-specific full-size antibody was evaluated as an alternative to protect plants from virus infection (Zimmermann et al., 1998). The TMV specific scFv accumulated to very low levels in the plant cytosol (0.00002% of total soluble protein). Nevertheless, the low cytosolic scFv accumulation led to remarkably enhanced resistance although the amounts of expressed scFv were approximately 20,000-fold lower when compared to elite plants secreting high levels of the TMV-specific full-size antibody (Zimmermann et al., 1998). Transgenic plants accumulating the TMV-specific scFv in the cytosol showed >90% reduction of local lesion number and a significant portion showed resistance in systemic infection assays. This phenomenon could be explained by the fact that upon infection only a few TMV molecules are needed to enter the cytosol of a plant cell to initiate viral replication and cell-to-cell movement of progeny RNA. Presumably, the low amount of intracellular expressed scFv is sufficient for neutralizing the invading virions either by interfering in viral uncoating or assembly of progeny virions.
A different approach for engineering disease resistant crops was developed by integrating antiviral antibody fragments in the plasma membrane in planta. TMV specific scFv fragments were efficiently targeted to the plasma membrane of tobacco cells by a heterologous mammalian transmembrane domain and the membrane anchored scFv fusion proteins, facing to the apoplast, retained antigen binding and specificity (Schillberg et al., 2000). Transgenic plants expressing membrane targeted scFv fusion proteins were resistant to TMV infection, demonstrating that membrane anchored anti-viral antibodies were functional in vivo, offering a powerful method to shield plant cells from invading pathogens.
The engineering of bacterial resistance is another interesting topic of commercial interest. Many different genetic strategies have been used to generate plants resistant to bacterial diseases, including enhancing natural plant defense, artificially inducing cell death at the site of infection and the expression of antibacterial proteins or peptides (Mourgues et al., 1998). Recently, it has been demonstrated that antibody-based resistance is also useful against bacterial diseases. Le Gall et al. (1998) have shown that expression of an scFv specific for the stolbur phytoplasma major membrane protein provided a way to control phytoplasma diseases. Stolbur phytoplasma are strictly restricted to the sieve tubes within the phloem tissues. Therefore, the phytoplasma specific scFvs were expressed through the secretory pathway. Transgenic tobacco shoots expressing phytoplasmaspecific scFv top-grafted on tobacco plants heavily infected by the phytoplasma grew free of symptoms while non-transgenic tobacco shoots showed severe stolbur symptoms.
These studies demonstrate the potential of heterologously expressed recombinant antibodies to protect plants against virus and bacteria. However none of these publication describe that Ab, rAb, rAb fragments for its own or Ab, rAb, rAb fragments fused to antifungal peptides or proteins expressed in plants can yield resistance against pathogenic fungi.
Pathogenic fungi are the most devastating of plant diseases and most challenging for antibody-based resistance approaches. Fungal infections affect crops by destroying plants and seeds and by contaminating the harvested crop with fungal toxins. When fungal pathogens infect plants, they parasitize the host plant for nutrients and their invasive mycelia spread throughout the host. The fungi secrete proteins essential for fungal pathogenesis as part of an intimate relationship between the host plant and pathogen for penetration and nutrition. These proteins and any others like surface structures are suitable targets that could be neutralized by expression of recombinant antibodies. The antibodies may have to be transported to the site of the infection since the fungi should be inactivated prior to degradation of the host cell wall. This could reduce the damage caused by fungi without the environmental pollution that occurs when fungicides are used to control such diseases.
In mammals, antibody-based protection against fungal pathogens has been shown. (Yuan et al., 1998). In plants, symptom development for avocado, mango, and banana infected with Colletotrichum gloeosporioide was delayed using polyclonal antibodies that bound fungal pectate lyase (Wattad et al., 1997). To date, there is no evidence that an antibody produced in transgenic plants can protect it from fungal infection. An attractive idea is to use antibodies as a carrier to target linked anti-fungal (poly)peptides to the fungal cell surface, where they destroy the hyphae. Moreover, antibodies may directly contribute to increase crop yield by neutralizing fungal toxins in infected crop harvests. A wide variety of fungal metabolites are toxic to plants, animals and humans, having dramatic effects on animal and human health, in addition to their involvement in plant pathogenesis (Desjardins & Hohn, 1997).